Summary I propose to develop and apply innovative structural biology and protein engineering tools to investigate the molecular mechanism of antigen editing and display on major histocompatibility complex (MHC or HLA in humans) molecules. The MHC encodes the most polymorphic proteins in the human genome, and is associated with more diseases than any other region. Unravelling the function of these proteins will help us understand autoimmune diseases, such as diabetes, multiple sclerosis and arthritis, and immune responses to viral pathogens and developing tumors. In particular, Class-I proteins encoded by the MHC (MHC-I) play a pivotal role in alerting the rest of the immune system to peptide antigens, derived from self-proteins, intracellular pathogens or tumors, by interacting with clonotypic T cell receptors (TCRs) expressed by cytotoxic CD8+ T cells. Key to the assembly of properly conformed MHC-I with bound peptide antigen are molecular chaperones that actively select high-affinity peptides for the displayed repertoire. Besides the basic science merit, characterizing this mechanism in atomic detail has important clinical implications, as suggested by immunodeficiencies resulting from dysregulation of the peptide-loading process, the downregulation of chaperone functions in tumors, and the direct targeting of chaperones by viral immune evasion strategies. Despite a large number of functional and structural studies, the use of conventional methods has proven ineffective for elucidating the 3D structure of the MHC-I/chaperone complex together with bound peptides. This is due to the highly dynamic nature of peptide interactions within the chaperoned MHC-I groove. As a result, the crucial conformational changes needed for antigen editing remain incompletely characterized. To remove these bottlenecks, I have developed a new methodology that combines complementary datasets from NMR and cryoEM with the computational modeling program, Rosetta, to obtain high-resolution structures of such challenging complexes. Here, I propose to apply this powerful integrative modeling approach to characterize chaperone complexes with human HLA molecules, and to elucidate dynamic transitions between peptide conformations which govern antigen editing. Our structural results will be further explored using protein directed evolution, and explicitly addressed in a cellular context using functional experiments, followed by a detailed proteomic analysis. As a long-term goal, I plan to use a structure- guided approach to engineer novel chaperone functions with custom specificities, to be used in emerging immunotherapy applications against graft rejection, autoimmune diseases, pathogen infection and cancer.